Various publications or patents may be referenced in this application in order to describe the state of the art to which the invention pertains. Complete citations for these references may be found at the end of the specification. Each of these publications or patents is incorporated by reference herein. The steroid and thyroid hormone superfamily of nuclear receptors in mammals and insects is composed of over 150 known proteins. These receptors fall into at least two functionally distinct categories (Class I and Class II) based on whether they function as homodimers or heterodimers, respectively (4). of the two classes, only the Class II receptors function in the nucleus as heterodimers to effect target gene(s) expression in the presence of hormone. The best studied examples of Class II receptor proteins are Retinoic Acid Receptor (RAR), Vitamin D Receptor (VDR), Thyroid Hormone Receptor (TR) and Retinoid X Receptor (RXR). Upon ligand binding, these receptors bind to the 5′ regulatory region of the target gene resulting in transcriptional activation (transactivation) of target genes. Gene expression occurs when the activated receptors and other transcription initiating factors act in concert. All nuclear receptors share a common multi-domain structure. They are divided into six domains that are termed A/B, C, D, E and F domains. These domains have been found to possess the following function: transcription activation function (A/B or AF-1, E or AF-2), a DNA binding (C), a steroid binding and dimerization (E), and a nuclear localization (D). The AF-1 domain is responsible for ligand-independent transcription activation, while the AF-2 domain has ligand-dependent activity. Both of these domains may act independently or in concert. For instance, removal of the AF-1 domain in the estrogen receptor has no effect on 17-estradiol (E2) induction of a reporter construct containing a vitellogenin estrogen response element (ERE), whereas the same AF-1 deficient ER demonstrates only 20% of wild-type induction of a pS2-ERE responsive element (18). Estrogen receptor studies using AF-1 and AF-2 truncated ER have demonstrated that AF-1 responds to growth factors that act via second messengers such as cAMP, whereas AF-2 is E2 ligand dependent (9). For the insect ecdysone receptor, removal of AF-1 domain of spruce budworm EcR did not affect either DNA or ligand binding activity of the EcR/USP heterodimer (14). In addition to the Class II receptor proteins found in mammals as described above, ecdysone receptor has been identified in Drosophila melanogaster (DmEcR), Bombyx mori (BmEcR), Manduca sexta (MsEcR), Chironomus tentans (CtEcR), Choristoneura fumiferana (CfEcR), and from mosquito Aedes aegypti (AaEcR) (See review in (12)). The ecdysone receptor (EcR) binds the steroid hormone 20-hydroxyecdysone and, when heterodimerized with the product of the ultraspiracle gene (USP), transactivates target gene expression. It has also been shown that EcR/USP heterodimerization is required for both DNA binding (20) and ligand binding (21), (13) in D. melanogaster and C. fumiferana. Additional chemical ligands besides 20-hydroxyecdysone, such as other hormone agonists, will also bind to these receptors and cause transactivation of a target gene. To date, the insect USP receptors have been cloned from D. melanogaster (DmUSP), B. mori (BmUSP), M. sexta (MsUSP), A. aegypti (AaUSP), and C. fumiferana (CfUSP) (See review in (13)). Mammalian retinoid X receptors (RXR) are homologues of insect USP. It has been shown that mammalian RXRs are capable of substituting for USP to form heterodimers with insect EcRs (19, 20; 21 and 22).
Ligand dependent transactivation systems have been reconstructed in yeast for mammalian receptors such as estrogen, thyroid hormone, androgen receptor etc. The coactivators such as GRIP1 and RIP140 have been used to enhance ligand dependent transactivation in yeast for thyroid hormone (Paul Walfish et al., (1997) PNAS 94:3697–3702) and estrogen receptor. Despite the fact that ligand dependent transactivation for series of mammalian nuclear receptors have been successfully reconstructed in the yeast, to date a ligand dependent system was not available for studying insect ecdysone receptors. Others have observed that transcription of reporter genes in the presence of ecdysone receptor expressed in the yeast is constitutive (De la Cruz and Mak, 1997).
One goal of the insecticide industry is the development of safe chemicals which are not only effective but also pest selective. It is an object of the present invention to provide a unique, yeast-based, ligand-mediated transactivation system to facilitate screening of new pesticide chemicals as well as to validate and improve upon potentially valuable insecticidal candidates.